The present invention relates to hollow fiber cartridge devices for in vitro cell growth or cell population expansion.
Hollow fiber cartridges or bioreactors for in vitro cell growth are well known in the art and are available from several commercial sources. These devices generally have a housing and a plurality of capillaries or hollow fiber membranes. The capillaries extend between an inflow opening at one end of the cartridge and an outflow opening at the other end. The capillaries have selectively permeable walls through which growth media or culture media, canning essential nutrients and gases, can diffuse. The interiors of the walls of the plurality of capillaries define a lumen extending between the inflow and outflow openings, and the outside of the capillaries and the housing define an extracapillary space (ECS) where cell growth or population expansion typically takes place. The housing generally includes one or two ports providing access to the ECS so that cells may be added or removed therefrom.
The cells are typically cultured in the ECS in growth media which originates from the lumen. The media in the lumen diffuses through the selectively permeable walls of the hollow fiber membranes into the ECS to stimulate the culture or growth of the cells. To cause media to move through the walls of the hollow fiber membranes into the ECS, growth media is typically pumped through the lumen of the cartridge.
As the media passes from the inflow opening at one end of the cartridge, through the lumen, and out the outflow opening at the other end of the cartridge, a pressure drop occurs from the inflow opening to the outflow opening. At the higher pressure inlet end of the cartridge, a radial convective flow of media moves from the lumen to the ECS bathing the cells in fresh nutrient-containing media. At the opposite end of the cartridge, near the outflow opening, media flows in the opposite direction, thereby removing metabolic waste products and other secreted cell products from the ECS and carrying them through the hollow fiber membranes and into the lumen. These products are then carried out of the cartridge through the outflow opening. Such devices are disclosed by Knazek et al. (U.S. Pat. Nos. 3,821,087; 3,883,393; and 4,220,725) and Yoshida et al. (U.S. Pat. No. 4,391,912).
In recent years, there has been an increasing demand for mammalian cell secreted products. Mammalian cell culture is now utilized to produce many important cell products for human use including monoclonal antibodies, vaccines, lymphokines, hormones, growth factors, enzymes, and other recombinant DNA products. As these products move from research and development, through clinical trials and to the market, a need for an economical large-scale method of production is required. The hollow fiber cell culture devices disclosed by Knazek et al. and Yoshida et al. are not suitable for large-scale manufacturing of mammalian cell secreted products.
These devices are of limited value for large-scale cell production and manufacturing of secreted cell products, particularly mammalian cell products, because of reasons related to the following: (1) the relatively large molecular weight cut-offs of the hollow fiber membranes which allows the required amount of oxygen to enter the ECS, but do not retain all secreted cell products or expensive serum-type nutrients; (2) inefficient oxygen diffusion to the ECS which limits cell growth; (3) formation of nutrient gradients which limit cell growth; and (4) formation of microenvironments for cell growth within the ECS which limits the full use of the entire capacity of the devices for cell growth.
The prior art devices utilize hollow fiber capillary membranes that have large enough molecular weight cut-offs to enable the secreted cell products to pass from the ECS to the lumen. This is undesirable for large-scale production because the secreted cell products become diluted in the large volumes of media necessary to maintain the cells. In addition, serum supplements to the media are generally required on the lumen side of the device. Serum addition to large volumes of media, which are necessary for large-scale production, can be very expensive and can also result in the extensive addition of impurities (e.g. contaminating proteins) to the already significantly diluted secreted cell products. These impurities can increase the cost of purifying the secreted products and often result in decreased yields.
If lower molecular weight cut-off hollow fiber capillaries are utilized in these devices, however, oxygen diffusion to the cells in the ECS will be severely limited. The smaller the cut-off, the less oxygen diffusion. The prior art devices often include a second type of hollow fiber capillary that is especially permeable to nutrients (see e.g. Knazek et al. U.S. Pat. Nos. 3,821,087; 3,883,393). This is also undesirable for large-scale production, because cells will preferentially grow on the nutrient source capillaries, not the oxygenation capillaries, thereby decreasing the surface area in the device utilized for cell growth. Furthermore, the inclusion of oxygenation capillaries in those devices, which generally have large molecular weight cut-offs (e.g. about 0.2 microns), does not allow for the retention of secreted cell products or other high molecular weight proteins in the ECS.
It is desirable to retain secreted cell products in the ECS in large-scale production systems, because this allows the secreted cell products to be concentrated in the ECS, rather than being concentrated after leaving the cartridge using one of several tedious or time consuming concentration procedures. It will be appreciated that highly concentrated products are less expensive to process and purify. It is also desirable to retain expensive high molecular weight proteins necessary for cell growth (e.g. serum, growth factors, hormones and cell secreted products) in the ECS. This would allow for the addition of these molecular species in relatively modest quantities to the ECS, rather than to the large lumenal media volume, which would require significantly larger quantities in order to provide the necessary concentrations.
The prior art hollow fiber devices are also limited in their use for large-scale production of secreted cell products by the formation of gradients in the ECS. When nutrient media is delivered via a motive force to the inflow opening of the hollow fiber device, the porous nature of the capillaries causes a change in hydrostatic pressure across the length of the cartridge. Cells at the inlet or high pressure end are continually exposed to a convective flow of fresh nutrients and oxygen, while cells at the outlet or low pressure end are continually exposed to a concentration of metabolic waste products from cells upstream and have limited access to fresh nutrients and oxygen. This results in the formation of a heterogeneous culture environment in the cell-occupied ECS due to the unequal nutrient distribution and the concentration of waste products. These nutrient gradients make it impractical to construct cartridges any longer than about 3-4 inches in length. Longer lengths only provide sufficient nutrients for cell growth in the high pressure inlet portion of the ECS.
Poor circulation of the media in the ECS of prior art devices can also lead to the formation of microenvironments having widely varying cell growth potentials. Microenvironments can occur when pockets of metabolically-active cells near the inlet end of the device secrete waste products into an immediately adjacent area. The waste products accumulate and are not quickly removed through the outlet end of the device. This accumulation of waste products results in microenvironments where cells are unable to grow. It will be appreciated that increasing the length of these devices simply increases the pressure drop and results in a worsening of the problems associated with the gradients and microenvironments created in the prior art devices. These problems severely limit the use to these devices for large-scale production of secreted cell products.
Knazek et al. (U.S. Pat. No. 4,184,922) disclose an improved device that decreases the microenvironment formation problem. This is accomplished by weaving together two separate perfusion circuits. By altering the pressure differences between the circuits, waste products can be removed more efficiently. However, this device still allows for the formation of gradients, which therefore make it unsuitable for large-scale production.
Cracauer et al. (U.S. Pat. No. 4,804,628) disclose an improved hollow fiber culture device that incorporates an external chamber in fluid communication with the ECS. By pressurizing the lumenal flow path, media flux to the ECS is increased. As media is forced into the ECS at the inlet end of the device, it subsequently is forced down the length of the cartridge to the outlet end. A percentage of the media moves into the expansion chamber through a unidirectional valve and a percentage exits across the capillaries to the lumen as in conventional devices. When the external chamber fills, the pressure in the chamber is increased to force the media back into the ECS through a second unidirectional valve which directs the media to the area of the ECS near the inlet end of the cartridge. This cycling helps mix the ECS media, thereby minimizing gradients and microenvironments which might otherwise exist. Oxygen diffusion is still limited, however, and gradients still occur.
The present invention is designed to overcome these and other limitations of the prior art devices and to provide a cell culture environment where cells are equally perfused with nutrients, and waste products are equally removed across the entire length of the hollow fiber cartridge.